Overview

An introduction to genetic inheritance is presented, including the principles of segregation and dominance.

Authors

• Don Lee, Department of Agronomy and Horticulture, University of Nebraska-Lincoln

Published 2003

Introduction - The Inheritance of Variation

The Ford Bronco came to a stop and generated a cloud of fine dust that blew 100 yards or so with the southern breeze into the traffic on Huntington Avenue. Regular travelers of that street taking a quick glance through the dust would see a routine play out that was familiar to them. Dr. Jim Specht, a geneticist at the University of Nebraska, would pile out of his Bronco with a notebook and head deliberately into a soybean field. Hours later spectators would see Dr. Specht in the same field. Perhaps he would be bending down for a closer look at a plant or pausing to write something in his notebook. “That guy spends hours out there, what’s he looking for?” they would wonder.

Genetic variation was what Dr. Specht’s keen eyes were diligently searching for. The field contained thousands of soybean plants that were the subjects of a carefully planned and implemented experiment designed to reveal some understanding about the control of traits in soybean (Fig. 1). The experimental design was a classical approach used by many scientists interested in understanding how variation in traits is controlled and inherited.

Experiment Outline

[latex]M_1[/latex]

An outline of Dr. Specht’s experiment is given below. (It is difficult to keep track of the details of this experiment so pull out some paper and make an outline of what was observed and done each generation)

1. He first obtained seeds from a line of soybeans that are true breeding. This means that when the soybean uses its normal self-pollination mode of sexual reproduction all of the resulting progeny will be identical to the parent plant.
2.  He carefully treated the seed with ethyl methane sulfate (EMS, Fig. 2). This chemical is a mutagen and will cause changes in the genetic makeup of the cells in the seed.
3.  He planted the mutagenized seed. The seed is called the [latex]M_1[/latex] seed for the first generation following the mutagenesis treatment. The plants generated are self-pollinated and make [latex]M_2[/latex] seed. Seed is harvested from the plants with a combine and put into one bag.

[latex]M_2[/latex]

4. Next, he planted the [latex]M_2[/latex] seed. This is the field of soybean plants Dr. Specht was seen spending lots of time in.
5. In June, July and August, Dr. Specht identified and tagged plants that had a different and interesting appearance or phenotype. At this stage Dr. Specht knew that the phenotype of any plant could be a result of variation in the plant’s genetic makeup or in the environment the plant happens to be growing in. (Dr. Specht was looking for plants that did not stop flowering or indeterminate plants. He never observed any but he did observe plants with other strange phenotypes.)
6. In October, Dr. Specht pulled the tagged plants, harvested the seed from a single plant, placing that seed in a labeled envelope. The seed was the [latex]M_3[/latex] generation. Notes were recorded in the computer so the phenotype of the plant that was the parent of each envelope of seeds could be looked up.

[latex]M_3[/latex]

7. The next spring Dr. Specht planted about 20-30 [latex]M_3[/latex] seeds from each envelope. This time seeds were planted in progeny rows (Fig. 3). Seeds from each envelope are planted in the same row and the row is carefully staked and mapped in the field.
8. Again that next summer Dr. Specht observed plant phenotypes and took notes. Dr. Specht was again frequently seen in these plots. Three kinds of rows of [latex]M_3[/latex] plants were observed (Fig. 4):
   1. 1. All the [latex]M_3[/latex] plants in the row looked normal, the unique phenotype of the [latex]M_2[/latex] parent was not passed on to the progeny.
      2.  All the [latex]M_3[/latex] plants in the row had the unique trait of the [latex]M_2[/latex] parent.
      3. Some of the plants in the row had the unique phenotype of the parent and others had the normal condition.

Explanations

Dr. Specht’s experiments continued but let’s stop here and evaluate his early results by asking a question. How do you explain the three types of rows observed in step #8?

1. The [latex]M_3[/latex] plants do not express the unique phenotype that was observed in the [latex]M_2[/latex]. The simplest explanation is that the unique phenotype was controlled by environmental influences, not by genetic variation in the [latex]M_2[/latex] plant. Therefore the phenotype was not inherited.
2.  The [latex]M_3[/latex] plants all expressed the unique phenotype observed in the [latex]M_2[/latex]. The unique trait is inherited. Furthermore, the [latex]M_2[/latex] plant was only able to pass this phenotype on and were therefore truebreeding for the unique trait. The genetic makeup of the [latex]M_2[/latex] parent of these [latex]M_3[/latex] plants must have been homozygous. The key to understanding this term is the prefix “homo” which means same. If the [latex]M_2[/latex] parent was homozygous and self-pollinates we would expect the [latex]M_3[/latex] progeny to be the same. (How could we test this?)
3.  Some of the [latex]M_3[/latex] plants have the unique phenotype of the [latex]M_2[/latex] parent but others express the normal condition of the original true breeding line. The [latex]M_2[/latex] plants that produced these types of rows must be heterozygous. The key to understanding this term is the prefix “hetero” which means different. Heterozygous parents are not true breeding.

Experiment Summary Statements

At this stage of Dr. Specht’s genetic experiment he could make these summary statements:

1. The mutagen applied to the true breeding seed induced phenotype variations in the soybeans. Dozens of unique phenotypes were discovered in the plots of [latex]M_2[/latex] plants while unique phenotypes would rarely be observed in the original true breeding soybean line.
2. The mutagen induced mutations or changes in the genetic makeup inside of the plants. These genetic changes were passed on to offspring and therefore the unique phenotypes were inherited.
3. The genetic makeup of the [latex]M_2[/latex] plants with mutations was not always the same. Some were homozygous and truebreeding and others were heterozygous.
4. The mutations were random. The original true breeding line was a determinant type soybean, which means it quit growing and producing flowers about mid-July. A mutation that causes a change in this phenotype to the indeterminate type which continues to grow and flower would be easy to see in the field. Despite his hours of observation, Dr. Specht never found this mutant and therefore could not study the genetic control of this trait. He did however observe some other interesting mutants. These phenotypes had never been reported in soybean. Therefore Dr Specht continued this experiment but took a different approach to study the inheritance of other traits.

The Monohybrid Cross Experiments of Dr. James Specht

The next round of experiments was designed to gain a more focused understanding of the genetic control of two of the mutant traits. Dr. Specht selected two mutants for these studies. One mutant grew normally at first and then began to develop lesions such that its leaves looked as if they had a pathogenic infection. This trait was called the disease lesion mutant.

The other mutant never grew normally but expressed an extreme dwarf phenotype. The plants are so small that it was surprising Dr. Specht even observed the original mutant plant in the field of [latex]M_2s[/latex].

The disease lesion and dwarf [latex]M_2[/latex] plants were both homozygous. What type of [latex]M_3[/latex] rows did they produce? They generated rows of progeny that all expressed their unique phenotypes. Some of these [latex]M_3[/latex] plants were then used in a monohybrid cross experiment.

Monohybrid Cross Experiment Outline

This experiment is called a monohybrid cross because Dr. Specht was tracking the inheritance of one trait that differed between the parents that were crossed. His monohybrid cross experiment was carried out as follows (Fig. 5):

1.  Cross a mutant plant with a truebreeding normal plant using the normal plant as the pollen donor. The seed produced is called [latex]F_1[/latex] seed (first filial or first family generation). Repeat this using the mutant as the male. This is the reciprocal cross. Again [latex]F_1[/latex] seed were produced, but kept separate from the first cross.
2.  Plant the [latex]F_1[/latex] seed and observe the phenotype. Allow the plants to self-pollinate to produce [latex]F_2[/latex] seed. Harvest the seeds from an individual plant and place into a labeled envelope. Do the same with the reciprocal cross.
3.  Plant the [latex]F_2[/latex] seeds from each envelope into a separate row. Observe and record the phenotypes of plants in each row. Allow the plants to self-pollinate to produce [latex]F_3[/latex] seed.
4.  Harvest the [latex]F_3[/latex] seed from each plant; place in a labeled envelope and record in the computer the phenotype of the parent for each envelope.
5. Plant the [latex]F_3[/latex] seed in progeny rows. Walk the rows (lots of rows in this generation!), score and record the phenotypes observed in each row.

Monohybrid Cross Experiment Data

The data collected in the monohybrid cross experiment for the disease lesion trait is given in Table 1. The data in Table 1 is summarized from a journal article by J. Chung, P.E. Staswick, G.L. Graef, D.S. Wysong, and J.E. Specht titled Inheritance of a disease lesion mimic mutant in soybean (1998 in the Journal of Heredity, 89:3).

Now Dr. Specht’s objective was to explain this data. He needed to formulate a hypothesis that would be supported by the results of the experiment. The best way to devise a hypothesis is to summarize the results of the experiment in simplest terms.

1. 1. All of the [latex]F_1[/latex] plants had the same phenotype, the normal trait.
   2. In both the first cross and the reciprocal cross, the mutant phenotype was not apparent in the [latex]F_1[/latex] but it reappeared in the [latex]F_2[/latex]. With both traits, the normal phenotype appeared in about [latex]\frac{3}{4}[/latex] of the [latex]F_2[/latex] and the dwarf or disease lesion mimic trait in [latex]\frac{1}{4}[/latex] of the [latex]F_2[/latex] (Fig. 6).
   3. [latex]F_2[/latex] plants with the mutant phenotype were all true breeding. [latex]F_3[/latex] progeny of mutant [latex]F_2s[/latex] all expressed the mutant trait.
   4. Two types of plants had the normal phenotype in the [latex]F_2[/latex]. About [latex]\frac{1}{3}[/latex] of the [latex]F_2[/latex] normals were true breeding and only produced [latex]F_3[/latex] progeny with the normal phenotype. The other [latex]\frac{2}{3}[/latex] of the normal [latex]F_2s[/latex] produced both normal and mutant [latex]F_3[/latex] offspring, again in a 3:1 ratio.

The Principle of Segregation

These results can be explained using the principle of segregation.

1. The traits variation observed was controlled by genes. These genes somehow store information in the cells of living things to direct the expression of traits. Genes can be changed to alternative forms called alleles.
2. Genes are found in pairs in somatic cells that make up the plant.
3. The paired genes separate during gamete formation. One gene from each pair goes into a gamete.
4. The gametes fuse at random during sexual reproduction, producing the next generation (seed in this case).

Segregation describes the behavior of something that Dr. Specht could not see as he observed plants in the field. He cannot prove this is happening with his experimental data but he can test the validity of the principle in explaining his results. That is what we will try to do.

Dr. Specht started with a true breeding line that was homozygous at all of its gene pairs. There was no genetic variation among progeny in this line. The mutagen created an alternative allele at a gene pair controlling leaf traits and an allele at a gene pair that controls the growth of the plant. Once a gene is altered and a new allele created, the new allele will be stable and pass on this altered information from cell to cell and parent to offspring.

Using Punnett Squares to Prove Segregation

We can use the letter symbol ‘D’ to represent the normal leaf growth allele and ‘d’ to represent that alternative version causing disease lesion mimics.

The homozygous normal parent had two copies of the ‘D’ allele. Therefore it had the genotype DD. The homozygous disease lesion mutant was the genotype dd. Therefore the first cross could be depicted as follows:

The paired genes separated when the parents made gametes. Since both parents were homozygous at this gene pair, they each made only one kind of gamete. When these gametes came together only one genotype combination was possible, Dd or the heterozygous genotype (Fig. 7).

The [latex]F_1[/latex] plants had the same appearance or phenotype as the normal parent but our hypothesis predicts that they carry the ‘d’ allele. If this is true they must mask its presence with the ‘D’ allele. Thus we are proposing that the normal leaf allele, ‘D’ is dominant over the recessive ‘d’ allele. The only way to test the hypothesis that the ‘d’ allele is present but masked in the [latex]F_1[/latex] is to obtain evidence that the [latex]F_1s[/latex] could pass the ‘d’ allele on to their progeny. The [latex]F_2[/latex] data confirmed the hypothesis.

The occurrence of the mutant phenotype among the [latex]F_2[/latex] progeny is evidence that the [latex]F_1[/latex] plants had the ‘d’ allele and passed it on. The 3:1 phenotype ratio observed in the [latex]F_2[/latex] is consistent with the principle of segregation (Fig. 8).

According to the hypothesis of segregation, all three possible genotypes should be produced in the [latex]F_2[/latex] generation. What should the genotypic ratio be? How can we determine a plants genotype? The Punnett square predicts a 1:2:1 genotype ratio for the DD, Dd, and dd [latex]F_2s[/latex]. Because we can observe phenotype and not genotype in this experiment, we need to attempt to indirectly verify this ratio. Again this can be accomplished by observing the [latex]F_3[/latex] progeny.

The data table gives the results of selfing the [latex]F_2s[/latex] with the normal phenotype. Two different types of normal [latex]F_2s[/latex] were again observed. Homozygous [latex]F_2s[/latex] would give rows of all normal [latex]F_3s[/latex] and heterozygous [latex]F_2s[/latex] gave mixed rows of both normal and mutant [latex]F_3s[/latex]. What ratio would we expect for these two types of rows? Again, the Punnett square predicts a 1:2 ratio. Do the results support this? Yes, the mixed rows were in the majority and if the numbers are combined from both reciprocal crosses, normal [latex]F_2[/latex] plants gave 37 [latex]F_3[/latex] rows that were all normal and 62 rows that were mixed. These numbers agree with what the principle of segregation predicts.

A Common Pattern

Dr. Specht’s experimental design was applied to the dwarf mutant as well and the same pattern of inheritance and genetic hypothesis could be applied. The recessive dwarf trait also appears to be controlled by segregation of a single gene pair in soybean. The results of this experiment are summarized in Table 1.

For a scientist, there are few things more satisfying than performing an experiment that provides new knowledge and gives results you can understand. Dr. Specht’s experiments revealed the presence of genes that play a critical role in leaf or plant development in the soybean. We cannot see these genes when we look at the soybean with the naked eye but proposing these genes exist and behave as described explains the inheritance patterns observed in these traits. If the genes are altered (by the EMS) they no longer control normal plant growth as they should. This is a common theme in classical genetics, genes can only be studied if they vary among individuals in a species. The discovery will not end here. Dr. Specht’s research group is currently mapping the gene to a specific position on a chromosome in the soybean. At the end of the research paper Dr. Specht also invites other researchers interested in cloning the disease lesion mutant gene to work with these plants. Researchers know that collectively, more is gained when knowledge is shared.

The sharing of ideas in genetics started over 100 years ago. In fact, the idea of how to organize and interpret the information from this classical genetic experiment in soybean was borrowed from a research paper published in 1865 by Gregor Mendel. The principle of segregation was a breakthrough idea in biology and Mendel was the first scientist to propose this from his experiments on peas.

Summary

While genetics has a lot of terminology, there are key principles that can be applied to explain the variation in trait inheritance observed in many organisms. The principles of segregation and the dominance of one allele over another can allow geneticists to understand and predict how traits are passed on.

Acknowledgements

Development of this lesson was supported in part by Cooperative State Research, Education, & Extension Service, U.S. Department of Agriculture under Agreement Number 98-EATP-1-0403 administered by Cornell University and the American Distance Education Consortium (ADEC). Any opinions, findings, conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture.